DNA Sequencing (Sanger Method) - Dideoxynucleotides (ddNTPs)

Dideoxynucleotide triphosphates (ddNTPs) lack the 3'-OH group of dNTPs that is essential for polymerase-mediated strand elongation in a PCR. Therefore, ddNTPs are used in combination with a modified Taq polymerase (e.g. JBS Sequencing polymerase or Thermosequenase™) as 3'-end chain terminators in Sanger sequencing[1] (Fig. 1) and single nucleotide polymorphisms (SNPs) genotyping by single base pair extension (SBE)[2] (Fig. 2). Our ddNTPs are > 98 % pure (HPLC) and functionally tested in chain termination sequencing.

Sequencing PolymerasedNTPs5'-fluorescently labeled PrimerSanger Sequencing

Figure 1: The enzymatic dideoxy chain termination sequencing method of Sanger[1] relies on the linear amplification of a single-stranded template DNA using a 5'-fluorescently labeled primer and a modified Taq polymerase. The synthesis of the complementary DNA strand starts at the specific priming site and ends with the incorporation of a chain-terminating ddNTP that is randomly introduced instead of its corresponding dNTP. By using the four different ddNTPs in four separate reaction vials, a set of extended primer strands terminated at each A, C, G, and T are obtained. When these fragments are separated on a suitable gel matrix the sequence information can be obtained from the migration order of the bands (bottom to top).

Make sure you also check out our DNA Cycle Sequencing Kit!

Sequencing PolymerasePrimerSBE

Figure 2: The single basepair extension (SBE) assay is a reliable method for the detection of a priori known locations of single nucleotide polymorphisms (SNPs)[2]. It relies on the extension of a primer, designed to bind one nucleotide upstream of the polymorphic spot, by a matching ddNTP. Subsequent detection of the incorporated ddNTP is performed by mass spectrometry of the extended primer, thus revealing the nucleotide base at that position on the template strand[3].


Selected References

[1] Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463.
[2] Syvänen et al. (1999) From gels to chips: Minisequencing primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mut. 13:10.
[3] Griffin et al. (2000) Single nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. Trends Biotechnol. 18 (2):77.